Transformed microorganism and process for producing D-aminoacylase

ABSTRACT

A transformed microorganism prepared by inserting into a host microorganism with zinc tolerance a D-aminoacylase-producing gene which selectively produces D-aminoacylase alone between D-aminoacylase and L-aminoacylase. A process comprising culturing the transformed microorganism in a culture medium containing zinc ion and obtaining D-aminoacylase from the culture at a high efficiency.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national-stage filing under 35 U.S.C. §371 ofPCT/JP00/03932, filed Jun. 15, 2000. This application claims priorityunder 35 U.S.C. §119 to JAPAN 11/170555, filed Jun. 17, 1999.

REFERENCE TO SEQUENCE LISTING

This application contains a sequence listing of nucleic acid and aminoacid sequences.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a transformed microorganism prepared byinserting into a zinc-tolerant microorganism a D-aminoacylase-producinggene which selectively produces D-aminoacylase alone betweenD-aminoacylase and L-aminoacylase, and a process for producingD-aminoacylase by utilizing the transformed microorganism.

2. Description of the Related Art

D-aminoacylase is an industrially useful enzyme for the production ofD-amino acids of high optical purity, which are used for the side chainsof antibiotics, peptide drugs and the like.

Chemical and Pharmaceutical Bulletin 26, 2698 (1978) disclosesPseudomonas sp. AAA6029 strain as a microorganism simultaneouslyproducing both D-aminoacylase and L-aminoacylase.

Japanese Patent Application Laid-open No. Sho-53-59092 disclosesactinomycetes, such as Streptomyces olibaceus S•6245. The use of thesemicroorganisms results in the simultaneous production of both opticalisomers of aminoacylase, D-aminoacylase and L-aminoacylase. While theseorganisms are capable of producing D-aminoacylase, it is necessary toseparate this enzyme from its optical isomer, L-aminoacylase. Thus,laborious and costly procedures are disadvantageously required for theseparation of the two.

Japanese Patent Application Laid-open No. Hei-1-5488 disclosesAlcaligenes denitrificans subsp. xylosoxydans M1-4 strain as amicroorganism that selectively produces D-aminoacylase alone.

If this bacterial strain is utilized, no laborious work is required forthe separation of D-aminoacylase from L-aminoacylase. However, thecapacity of this bacterial strain to produce D-aminoacylase isinsufficient. Furthermore, the nucleotide sequence of theD-aminoacylase-producing gene is not elucidated in Japanese PatentApplication Laid-open No. Hei-1-5488. Thus, this document does notdescribe how to modify the D-aminoacylase gene so as to improve itsD-aminoacylase-producing capacity or describe the creation of atransformed bacterium with an ability to produce higher amounts ofD-aminoacylase.

BRIEF SUMMARY OF THE INVENTION

In view of the above, the present inventors Moriguchi, et al. elucidatedthe structure of the D-aminoacylase-producing gene in the Alcaligenesxylosoxydans subsp. xylosoxydans A-6 strain and demonstrated itsnucleotide sequence, which appears as SEQ ID NO: 1 in the sequencelisting. Further, it was found that genetic modification of theD-aminoacylase-producing gene successfully improved theD-aminoacylase-producing capacity of the resulting transformed bacterium(Protein Expression and Purification 7, 395-399 (1996)).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically depicts the plasmid used for ligation with theD-aminoacylase-producing gene.

FIG. 2 schematically depicts the plasmid ligated with theD-aminoacylase-producing gene.

DETAILED DESCRIPTION OF THE INVENTION

The inventors' subsequent research works have elucidated that theD-aminoacylase-producing capacities of various transformed bacteria withthe aforementioned D-aminoacylase-producing gene inserted therein aregreatly improved in zinc ion-containing culture media. It has also beenfound that the D-aminoacylase producing capacity of a transformedbacterium is prominently improved by controlling the zinc ionconcentration within a predetermined range.

Furthermore, it has been found that the above-mentioned effect variessignificantly depending on the type of a host microorganism and that ahost microorganism with high such effect generally exerts zinc toleranceeven prior to the transformation thereof. Herein, the term “zinctolerance” means that the growth potency of a bacterium as measured onthe basis of the cell weight (A660 nm) is hardly inhibited by theaddition of zinc ion.

The findings mentioned above indicate the following two points: (1) Theexpression of a transformed microorganism with aD-aminoacylase-producing gene of SEQ ID NO: 1 is enhanced in thepresence of a given quantity of zinc ion, though the reason has not beenelucidated. (2) Since it is believed that zinc ion functions in aninhibiting manner on common microorganisms, a congenitally zinc tolerantmicroorganism should be selected as a host to insert the gene therein soas to sufficiently procure the effect of zinc ion.

Based on the above-mentioned points, the invention provides amicroorganism transformed with a D-aminoacylase-producing gene, theD-aminoacylase-producing capacity of which can be greatly enhanced withthe addition of zinc ion to a culture medium therefor. The inventionfurther provides a process for producing D-aminoacylase using thetransformed microorganism.

The transformed microorganism of the invention is a microorganism havingacquired high-expression ability to produce D-aminoacylase in a zincion-containing culture medium. This transformed organism may be preparedby inserting a D-aminoacylase-producing gene into a zinc tolerant hostmicroorganism wherein the expression of a gene product of the insertedgene is enhanced in the presence of zinc ion. The transformedmicroorganism is a microorganism transformed with aD-aminoacylase-producing gene, and due to the addition of zinc ion tothe culture medium, the D-aminoacylase-producing potency thereof can beenhanced to maximum.

In the transformed microorganism of the invention, theD-aminoacylase-producing gene more preferably has a nucleotide sequenceof SEQ ID NO: 1 or a nucleotide sequence hybridizing to the nucleotidesequence of SEQ ID NO: 1 under stringent conditions and effectivelyencoding D-aminoacylase. It has been confirmed that aD-aminoacylase-producing gene having a nucleotide sequence of SEQ ID NO:1 is a gene the expression of a gene product of which can greatly beenhanced in the presence of zinc ion. Further, a gene of a nucleotidesequence hybridizing to the nucleotide sequence of SEQ ID NO: 1 understringent conditions and effectively encoding D-aminoacylase can beexpected to have similar characteristics.

More preferably, in the transformed microorganism of the invention, thehost microorganism is Escherichia coli. It has been confirmed thatEscherichia coli has zinc tolerance. Further, the mycological andphysiological properties, culture conditions and maintenance conditionsof Escherichia coli are well known. Thus, the production ofD-aminoacylase at high efficiency can be done under readily controllableconditions.

Still more preferably, in the transformed microorganism of theinvention, a D-aminoacylase-producing gene which is to be inserted intoa host microorganism is subjected to at least one of the followingmodifications (1) and/or (2). (1) A modification for improving thetranslation efficiency, comprising designing a specific nucleotidesequence (GAAGGA) (SEQ ID NO: 3) in the ribosome-binding site andinserting the nucleotide sequence in the position of the ninth baseupstream of the translation initiation point of the gene. Thismodification improves the translation efficiency of theD-aminoacylase-producing gene. (2) A modification for improving the geneexpression efficiency, comprising creating a HindIII recognition site ofEscherichia coli in the upstream and downstream of the gene,subsequently purifying and excising the resulting gene, and ligating thegene into an expression vector. This modification improves theexpression efficiency of the D-aminoacylase-producing gene.

A zinc-tolerant microorganism is used as a host microorganism forobtaining a transformed microorganism in accordance with the invention.More specifically, a microorganism should be used, the growth potency ofwhich in culture media, as measured on the basis of increase or decreaseof the cell weight (A660 nm), is not significantly inhibited by theaddition of zinc ion. Zinc tolerance may be evaluated by comparing thecell weight of microorganisms grown in a zinc-free culture medium withthe cell weight of the same microorganism grown in a medium containingzinc. On the basis of the cell weight (A660 nm) of the microorganism ina zinc-free culture medium, the cell weight in the same culture mediumunder the same conditions except for the addition of 2 mM zinc eitherincreases, or decreases within a range of 10%. Otherwise, theabove-mentioned cell weight in the same culture medium under the sameconditions except for the addition of 5 mM zinc increases, or decreaseswithin a range of 20%.

Although the taxonomical group of the host microorganism is not limited,it is generally preferable to use such host microorganisms that havewell known morphological and physiological properties and for which theculture conditions and maintenance conditions are also well known. Apreferable example of such a host microorganism is Escherichia coli.Compared with Escherichia coli, microorganisms of the speciesAlcaligenes xylosoxidans including A-6 strain do not have zinctolerance.

The means for inserting a D-aminoacylase-producing gene into a hostmicroorganism is not specifically limited. For example, aD-aminoacylase-producing gene may be inserted into either a plasmid or abacteriophage by ligation to plasmid or bacteriophage DNA.

The D-aminoacylase-producing gene in accordance with the invention is agene selectively producing D-aminoacylase as opposed to producing bothD-aminoacylase and L-aminoacylase. This gene is of a type in which theactivity expression is enhanced in the presence of zinc ion in theculture medium. As a preferable example of such D-aminoacylase-producinggene, the gene with the nucleotide sequence of SEQ ID NO: 1 has beenconfirmed. Further, genes of nucleotide sequences hybridizing to thenucleotide sequence of SEQ ID NO: 1 under stringent conditions andeffectively encoding D-aminoacylase are also preferable, except forgenes which do not actually enhance the activity expression with zincion in the culture medium.

The D-aminoacylase-producing gene with the nucleotide sequence of SEQ IDNO: 1 was obtained from the Alcaligenes xylosoxidans subsp. xylosoxidansA-6 strain. The A-6 strain is a D-aminoacylase-producing strain obtainedfrom soil in nature via screening.

The process for producing D-aminoacylase in accordance with theinvention comprises culturing any transformed microorganism as describedabove in a culture medium containing zinc ion, and obtainingD-aminoacylase from the culture. Zinc ion can be provided by adding anappropriate amount of a zinc compound, such as zinc chloride and zincsulfate, to the culture medium. This process enables the production ofD-aminoacylase at a high efficiency.

In the process for producing D-aminoacylase in accordance with theinvention, the concentration of zinc ion contained in the culture mediumis preferably controlled to be in the range of 0.1 to 10 mM. Thisprocess enables to optimize the zinc ion concentration in the culturemedium, and to produce D-aminoacylase at a particularly high efficiency.

In the process for producing D-aminoacylase, other procedures andconditions for carrying out the process are not specifically limited.Never the less, the culture is preferably carried out in a nutritiousculture medium containing tac promoter-inducing substances (for example,isopropyl thiogalactoside (IPTG), lactose and the like) as inducers.Further, the concentration of lactose then is preferably adjusted toabout 0.1 to 1%.

Best Mode for Carrying out the Invention

The best mode for carrying out the invention is described below inconjunction with a comparative example. The invention is not limited tothe best mode for carrying out the invention.

Obtaining the D-Aminoacylase Gene and Determining its NucleotideSequence.

The chromosomal DNA obtained from Alcaligenes xylosoxidans subsp.xylosxidans A-6 strain was partially digested with restrictionendonuclease Sau3AI, to obtain by fractionation DNA fragments of 2 to 9Kb. The resulting DNA fragments were inserted in and ligated into theBamHI recognition site of a known plasmid, pUC118. Escherichia coliJM109was transformed with the ligated plasmid to obtain anampicillin-resistant transformant strain. Among the thus obtainedtransformant strains, a strain with the ability to selectively produceD-aminoacylase alone was obtained. The transformant strain with thisability contained a plasmid with a 5.8-Kb insert fragment.

The 5.8-Kb insert fragment in the plasmid was trimmed down to deduce theposition of the D-aminoacylase-producing gene. According to generalmethods, then, the nucleotide sequence as shown in SEQ ID NO:1 wasdetermined for the DNA of about 2.0 Kb. An amino acid sequencecorresponding to the nucleotide sequence is also shown in the sequencelisting. Consequently, an open reading frame (ORF) consisting of 1452nucleotides starting from ATG was confirmed.

Modification of the D-aminoacylase Gene

From the plasmid with the 5.8-Kb insert fragment was excised a 4-Kb DNAfragment via BamHI-HindIII digestion, which was then ligated into aknown plasmid pUC118 to construct a ligated plasmid pAND118. Using theresulting plasmid, site-directed mutagenesis using primers was effected,to thereby prepare a ribosome-binding site (RBS)-modified plasmidpANSD1.

Using the plasmid pANSD1 as template, site-directed mutagenesis usingprimers was effected, thereby to prepare a plasmid pANSD1HE having anEcoRI recognition site and a HindIII recognition site immediatelyupstream the RBS and immediately downstream the ORF, respectively.

Then, the plasmid pANSD1HE was digested with restriction endonucleasesEcoRI and HindIII to prepare a 1.8-Kb DNA fragment, which was insertedin and ligated at the EcoRI-HindIII site in the plasmid pKK223-3 shownin FIG. 1 to obtain the plasmid pKNSD2 shown in FIG. 2.

Transformation of Escherichia coli with the D-Aminoacylase Gene

The plasmid DNA was inserted into a host strain derived from theEscherichia coli K-12 strain by the D. HANAHAN's method (DNA Cloning,Vol. 1, 109-136, 1985), thereby to obtain a transformed Escherichia coli(E. coli) TG1/pKNSD2.

Zinc-Tolerance of the Bacterial Strain from which D-Aminoacylase Genewas Obtained

The Alcaligenes xylosoxidans subsp. xylosoxidans A-6 strain was culturedat 30° C. for 24 hours in a culture medium (pH 7.2, zinc-free)containing 0.2% potassium dihydrogen phosphate, 0.2% dipotassiumhydrogen phosphate, 2% polypeptone, 0.01% magnesium sulfate and 1%glycerin, and in culture media of the same composition but with additionof zinc oxide to concentrations 0.2 mM, 2.0 mM and 5.0 mM, respectively.After culturing, the cell weight (A660 nm) was measured to evaluate thezinc tolerance. Then, the pH of the culture media after culturing wasmeasured. The results are shown in the column of “A-6 bacteria” in Table1.

TABLE 1 Zinc Cell concentration Post- weight Relative value Microbialstrain (mM) culture pH (A660) (%) A-6 bacteria 0.0 7.58 8.09 100.0 0.27.62 7.75 95.8 2.0 7.56 5.23 64.6 5.0 7.68 3.34 41.3 TG1 0.0 5.01 5.68100.0 (host bacterium) 0.2 4.99 5.93 104.4 2.0 4.98 5.55 97.7 5.0 5.014.98 87.7 pKNSD2/TG1 0.0 5.00 6.45 100.0 (recombinant 0.2 5.01 6.70103.9 bacterium) 2.0 4.98 6.09 94.4 5.0 5.01 5.47 84.8

Table 1 shows that the cell weight of the A-6 strain in the zinc-addedculture media was greatly decreased (decreased by about 35% in the 2.0mM zinc-added culture medium and by about 60% in the 5.0 mM zinc-addedculture medium), compared with the cell weight of the A-6 strain in thezinc-free culture medium. This indicates that the A-6 strain was notzinc-tolerant.

Zinc Tolerance of Host Bacterium

The zinc tolerance of the strain derived from the Escherichia coli K-12strain used as the host bacterium was examined, using a culture mediumof the same composition as for the A-6 strain, by measuring the cellweight (A660 nm) in the same manner. The results are shown in the columnof “TG1 (host bacterium)”.

Table 1 shows that the cell weight of the host bacterium in thezinc-added culture media was not so greatly decreased (decreased byabout 3% in the 2.0 mM zinc-added culture medium and by about 12% in the5.0 mM zinc-added culture medium, and even increased in the 0.2mMzinc-added culture medium), compared with the cell weight of the hostbacterium in the zinc-free culture medium. This indicates that the hostbacterium was zinc-tolerant.

Zinc Tolerance of Transformed Escherichia coli

The zinc tolerance of the transformed Escherichia coli (E. coli)TG1/pKNSD2 was examined using a culture medium of the same compositionas for the A-6 strain by measuring the cell weight (A660 nm) in the samemanner. The results are shown in the column of “pKNSD2/TG1 (recombinantbacterium)”.

Table 1 shows that the cell weight of the transformed bacterium in thezinc-added culture media was not so greatly decreased (decreased byabout 5% in the 2.0 mM zinc-added culture medium and by about 15% in the5.0 mM zinc-added culture medium, and even increased in the 0.2 mMzinc-added culture medium), compared with the cell weight of thetransformed bacterium in the zinc-free culture medium. This indicatesthat the transformed Escherichia coli was zinc-tolerant.

(Effect of Zinc Addition on Transformed Escherichia coli)

The transformed Escherichia coli (E. coli) TG1/pKNSD2 was pre-culturedin a culture medium (pH 7.0) containing 1% bactotryptone, 0.5%bacto-yeast extract, 0.5% sodium chloride and 100 μg/ml ampicillin, at30° C. for 16 hours.

Subsequently, the post-preculture transformed Escherichia coli wascultured at 30° C. for 24 hours in a culture medium (pH7.0, zinc-free)containing 0.2% potassium dihydrogen phosphate, 0.2% dipotassiumhydrogen phosphate, 2% polypeptone, 0.01% magnesium sulfate, 1% glycerinand 0.1% lactose as an inducer, and culture media of the samecomposition but with addition of zinc oxide to concentrations 0.2 mM and2.0 mM. Additionally, the broth-out pH of the culture broth as well asthe enzyme activity (U/mL) of D-aminoacylase in the culture broth (A660nm) was measured.

Consequently, the enzyme activity in the 0.2 mM zinc-added culturemedium was 58.85 U/mL (broth-out pH of 5.03) and the enzyme activity inthe 2.0 mM zinc-added culture medium was 109.79 U/mL (broth-out pH of5.11), compared with the enzyme activity of 21.78 U/mL in the zinc-freeculture medium (broth-out pH of 5.05). Thus, it has been confirmed thatthe addition of zinc ion, at least within a predetermined concentrationrange, greatly improves the D-aminoacylase-producing potency.

For comparison, additionally, the A-6 strain was pre-cultured in theculture medium for preculture (no ampicillin was however added) underthe same conditions, and was then cultured in the culture medium of thesame composition for culture, except for the change of the inducer from0.1% of lactose to 0.1% of N-acetyl-D, L-leucine. Then, the broth-out pHof the culture broth as well as the enzyme activity (U/mL) ofD-aminoacylase in the culture broth (A660 nm) was assayed.

Consequently, the enzyme activity in the 0.2 mm zinc-added culturemedium was 0.12 U/ml (broth-out pH of 7.48) and the enzyme activity inthe 2.0 mm zinc-added culture medium was 0.29 U/mL (broth-out pH of7.43), compared with the enzyme activity of 029 U/ML in the zinc-freeculture medium (broth-out pH of 7.47). Thus, no effect of zinc ionaddition on the improvement of the D-aminoacylase-producing potencycould be comfirmed.

INDUSTRIAL APPLICABILITY

As described above, D-aminoacylase, as an industrially useful enzyme,can be produced highly efficiently and selectively by using thetransformed microorganism of the invention.

1. An isolated microorganism comprising a nucleic acid sequence thatencodes the amino acid sequence of SEQ ID NO: 2, or a nucleic acidsequence from Alcaligenes, which encodes D-aminoacylase, which comprisesthe following sequence of restriction sites: Sal I, Bgl II and Pvu II,wherein said nucleic acid sequences comprise SEQ ID NO:3 in the ninthposition upstream from the first nucleotide in the start codon; saidmicroorganism is zinc resistant, and wherein the activity of D-aminoacylase encoded by said nucleic acid sequence in said microorganism isenhanced in the presence of zinc ion.
 2. The isolated microorganism ofclaim 1 that comprises a nucleic acid sequence that encodes the aminoacid sequence of SEQ ID NO:2.
 3. The isolated microorganism of claim 1that comprises the nucleic acid sequence of SEQ ID NO:
 1. 4. Theisolated microorganism of claim 1 that comprises a nucleic acid fromAlcaligenes which encodes D-aminoacylase, the activity of which isenhanced in the presence of zinc ion, wherein said nucleic acidcomprises the following sequence of restriction sites Sal I, Bgl II andPvu II, and comprises SEQ ID NO:3 in the ninth position upstream fromthe first nucleotide in the start codon.
 5. The isolated microorganismof claim 1, wherein the aminoacylase encoding nucleic acid sequence isobtained from Alcaligenes xylosoxidans, subsp. xylosoxidans strain A-6.6. The isolated microorganism of claim 1, wherein the aminoacylaseencoding nucleic acid sequence is modified by: creating a Hind IIIrecognition site upstream and downstream from the D-aminoacylase gene,excising or purifying the resulting modified gene and ligating themodified gene into an expression vector.
 7. The isolated microorganismof claim 1, wherein the zinc resistance of the host microorganism issuch that the cell weight of the microorganism either increases, ordecreases, within a range of 10% in a culture medium with 2 mM zincadded thereto on the basis of the cell weight measured at A660 nm in azinc-free culture medium.
 8. The isolated microorganism of claim 1,wherein the zinc resistance of the microorganism is such that the cellweight of the microorganism either increases, or decreases, within arange of 20% in a culture medium with 5 mM zinc added thereto on thebasis of the cell weight measured at A660 nm in a zinc-free culturemedium.
 9. The isolated microorganism of claim 1, which is Escherichiacoli.
 10. A process for producing D-aminoacylase comprising: culturingthe isolated microorganism of claim 1 in a culture medium containingzinc and recovering D-aminoacylase.
 11. The process of claim 10, furthercomprising culturing said microorganism in a medium containing a tacpromoter-inducing substance.
 12. The process of claim 10, wherein saidpromoter-inducing substance is isopropyl thiogalactoside (IPTG) orlactose.
 13. The process of claim 10, wherein said culture medium has aconcentration of lactose ranging from 0.1 to 1%.
 14. An isolated nucleicacid sequence which encodes the amino acid sequence of SEQ ID NO: 2, orwhich encodes a D-aminoacylase from Alcaligenes, which comprises thefollowing sequence of restriction sites: Sal I, Bgl II and Pvu II,wherein said isolated nucleic acid sequence comprises an upstreamribosome binding site comprising GAAGGA (SEQ ID NO: 3) in the ninthposition upstream from the first nucleotide in the start codon.
 15. Theisolated nucleic acid sequence of claim 14, which encodes the amino acidsequence of SEQ ID NO:2.
 16. The isolated nucleic acid sequence of claim14, further comprising an EcoR I site before said Sal I site and a HindIII site after the Pvu II site.
 17. A vector comprising the nucleic acidsequence of claim
 14. 18. An isolated nucleic acid sequence fromAlcaligenes that encodes a D-aminoacylase and which comprises thefollowing sequence of restriction sites: Sal I, Bgl II and Pvu II,wherein said isolated nucleic acid sequence comprises an upstreamribosome binding site comprising GAAGGA (SEQ ID NO: 3) in the ninthposition upstream from the first nucleotide in the start codon.
 19. Avector comprising the nucleic acid sequence of claim
 18. 20. Azinc-resistant host cell comprising the nucleic acid sequence of claim18.